Origin and genetic variability of populations of the invasive plant Rumex alpinus L. in the Giant (Krkonoše) Mountains

Abstract Monk's rhubarb, Rumex alpinus L. (R. alpinus), is a perennial plant native to the mountains of Central and Southern Europe. Currently, the distribution of R. alpinus has been partly affected by its utilization as a vegetable and a medicinal herb. In the mountains of the Czech Republic, it is considered an invasive plant, probably introduced into the Krkonoše Mountains by colonists from the Alps. This study's main aim was to verify whether R. alpinus was introduced into the Krkonoše Mountains by alpine colonists or whether it was anthropogenically introduced from the Carpathians. Furthermore, the genetic structure of native and introduced populations of R. alpinus was determined. For the evaluation of genetic structure, 417 samples of R. alpinus were collected from the Alps, Carpathians, Balkan, Pyrenees, and Czech Mountains. In total, 12 simple sequence repeat (SSR) markers were applied. The results of AMOVA showed a high 60% variation within populations, 27% variation among groups, and 13% among the population within groups. The overall unbiased gene diversity was high (^ĥ = 0.55). The higher level of genetic differentiation among populations (FST = 0.35; p < .01) indicated restricted gene flow between populations. Compared to native populations, limited genetic variability was observed in the nonnative populations. It was concluded that local adaptation, low gene exchange, and genetic drift affected the genetic diversity of nonnative R. alpinus. The results support a genetic link between Alpine and Czech genotypes of R. alpinus, while the Carpathians genotypes corresponded to the Balkan genotype.


| INTRODUC TI ON
. It is an anemophilous plant (Kubát, 1990); however, the pollen produced by flowering plants attracts numerous pollen-feeding insects, thereby taking part in the gene flow of R. alpinus populations (Klimeš, 1994;Št'astná et al., 2010). The production of seeds is very high (Št'astná et al., 2010); a flowering plant can produce approximately 11,500 seeds m−2 (Klimeš, 1992). The seeds can remain dormant for many years (Bucharová, 2003), and below the stand is a wealthy seed bank (Št'astná et al., 2010). The seeds are spread over long distances (100 m), mainly downstream, allowing the colonization of new habitats (Červenková & Münzbergová, 2009). Moreover, R. alpinus is also a clonal plant that reproduces through rhizomes (Klimeš, 1992), and the growth rate of populations is high and fast (Klimeš et al., 1993).
Whether a plant is native or nonnative in a given area is often difficult to determine. The allochthonous origin of many archaeophytes, epoecophytes, and ephemerophytes, e.g., Ballota nigra L.
Subsp. Nigra and Verbena officinalis L., is associated only with anthropogenically influenced communities (Kopecký, 1973;Pyšek et al., 2012). The distribution of some of them, such as Lamium album L. and Chenopodium bonus-henricus L., precisely defined the area of the original Czech agricultural settlement (Kopecký, 1973). However, the connection of a species to communities of anthropogenic origin may not provide adequate evidence for its allochthonous origin (Chytrý et al., 2005;Kopecký, 1973;Pyšek et al., 2012). According to Lokvenc (1978), this is the case for the species that have been previously collected as medicinal herbs, and they were also grown in gardens (Angelica archangelica L.). Therefore, it is difficult to determine the origin of some species that may have been grown as medicinal or valuable plants in the past.
The literature on the historical colonization of the Úpa and Elbe valleys in the Krkonoše Mountains by settlers from the Alps and their introduction of R. alpinus is well-established (Hendrych, 2001;Kopecký, 1973;Kubát, 1990;Lokvenc, 1978). Moreover, in the past decade, Professor Klimeš has been investigating the genetic origins of the Krkonoše settlers. His research has revealed that these ancestors hailed from sites in Styria's Salzkammergut region and South Tyrol in Austria and Italy (Klimeš, 2011). Whether R. alpinus is truly nonnative in the Krkonoše Mountains may not be certain. In Poland, among others, besides the Carpathian Mountains, where R. alpinus is a native plant (Klimeš, 1992;Stachurska-Swakoń, 2009), there is also a part of the Giant Mountains (Karkonosze), and according to Kwiatkowski (2003), R. alpinus is a native plant in Poland. Based on this statement and based on the available information, we decided to verify the origin of R. alpinus using SSR markers, because the possibility of a different origin of R. alpinus populations found in the Giant Mountains is considered. And we pose the following hypotheses: (i) Rumex alpinus, whose European distribution is determined by human activity, was introduced into the Czech part of the This is the first study focusing on the genetic variability and population structure of the problematic weedy plant R. alpinus, which could provide new assessments of this species under a genetic context and produce valuable data for further control and management of plant invasions.  (Kopecký, 1973;Kubát, 1990;Lokvenc, 1978;Pyšek et al., 2012;Št'astná et al., 2010), overall 88 plant samples were collected (Table 1 and Figure 1).

| Microsatellite (SSR) analysis
Based on the test, 12 polymorphic primer pairs were selected from the 15 primer pairs according to Šurinová et al. (2018) and used (Table S1). DNA amplification was performed in 5 μL reac-

| Statistical analysis
Analysis of the molecular variance test (AMOVA) with 1000 permutations was calculated in ARLEQUIN software ver. 3.5.2 (Excoffier & Lischer, 2010). The degree of genetic differentiation among populations was also evaluated using ARLEQUIN software using the distance matrix based on the fixation index (FST) generated by the program. Further, distance matrix based on geographical distances was calculated for R. alpinus populations within R program (R Core Team, 2020) version 4.0.3 using the routines in geosphere (Hijmans, 2021) library. These were subsequently logarithmically transformed and correlated with FST distance matrix using the Mantel test and 9999 permutations.
Nei's genetic distance was employed to obtain a UPGMA dendrogram after 1000 bootstrap samplings in TFPGA software (Miller, 1997).
The diversity indices for each population included the percentage of polymorphic loci, the average diversity of the loci using Nei's unbiased gene diversity ĥ (Nei, 1973), and the Shannon information index (Lewontin, 1972;Shannon & Weaver, 1949) were calculated using the POPGENE, version 1.32 (Yeh et al., 1999).
To assess the Hardy-Weinberg equilibrium, we used the ARLEQUIN software ver. 3.5.2 (Excoffier & Lischer, 2010). Was conducted an exact test using a Markov chain with a forecasted chain length of 1,000,000 and 100,000 dememorization steps (Guo & Thompson, 1992). Deviation from HWE was assessed at a significance level of p < .05. The results were interpreted according to established guidelines (Levene, 1949).
Another approach to studying population structure analysis is based on Bayesian statistics STRUCTURE, version 2.3.4 (Pritchard et al., 2000) was used to determine the genetic architecture of the R. alpinus populations. Ten independent runs of 1-20 groups (K = 1-20) were performed using the locprior model with admixture and correlated allele frequency (Falush et al., 2003;Hubisz et al., 2009) with the recommended 2,00,000 Markov chain iterations after a burn-in period of 1,00,000 iterations. The optimal value of K was estimated based on ln (K) and on the ΔK calculation, which considers the rate of change in the ln P (D) values among successive K runs to account for patterns of dispersal that are not homogeneous among populations (Evanno et al., 2005). The number (K) of clusters into which the sample data (X) were fitted with posterior probability Pr (X|K) was estimated using the same model with 1,000,000 Markov chain iterations after a burn-in period of 1,00,000 iterations (Evanno et al., 2005).
An exact test for population differentiation was calculated using the Tools for Population Genetic Analyses (TFPGA; version 1.3; Miller, 1997) with 1,00,000 recommended permutation steps.
To identify potential bottleneck events in the populations under investigation, we employed BOTTLENECK 1.2.02 software (Cornuet & Luikart, 1996;Piry et al., 1999) and heterozygosity excess resulting from population reduction was examined. We utilized three models of mutational equilibrium: the infinite allele model (IAM), the stepwise mutation model (SMM), and the two-phase mutation model (TPM), with the latter being the most appropriate for microsatellites.
For the TPM, we employed the default settings, which assumed that 70% of mutations occur in a single step, with a variance of 30 among multiple steps. The significance of these models was assessed using a one-tailed Wilcoxon rank test, which is suitable for datasets analysis with less than 20 microsatellite loci (Piry et al., 1999). A population was deemed to have experienced a bottleneck event only if all three models produced significant results (p-value ≤.05).

| RE SULTS
The number of alleles in different loci is presented in Figure S1, and the sum of all alleles for each population is in Table 2. In the 417 analyzed individuals, 146 alleles were identified for the 12 microsatellite loci (Table S3) with an average of 9.93 polymorphic loci ( Table 2). The mean of alleles per locus for all populations was 3 and ranged from the population of Eagle Mountains (1.5) to Carpathian Bihor Mountains population (5.2), where the highest number of nine alleles per locus was identified ( Figure S1). While the percentage of polymorphic loci was the highest in the Zakopane Lejowa glade population (100%) the lowest number was found in the Eagle Mountains (42%) ( Table 2). A total of 340 multilocus genotypes from 417 individuals of R. alpinus were identified. Some populations consisted partly or totally from identical clones, especially the Eagle Mountains population (Table S3) Table 2).
The overall mean value of I was 0.78 when all populations were included ( Table 2) Table 3).
Genetic variability was measured as the amount of observed or expected heterozygosity, presented in Table S2.  suggested that a significant proportion (71.7%) of the genetic variation can be explained by differences within populations, while a smaller 28.1% of differences can be explained by the variability among groups (mountains). As only 0.2% of the genetic variation was found among populations within groups, indicating only minor genetic differences among the populations within each group ( Table 6). Σ_polymorphic loci, the total number of loci in a population that has more than one allele; Σ-alleles, the total number of different alleles observed across all loci in a population.

TA B L E 2 (Continued)
Mountains (  Abbreviations: ĥ, observed heterozygosity; I, Shannon-Wiener Diversity Index; P (%), the percentage of polymorphic loci; St. Dev, standard deviation; Σ_polymorphic loci, the total number of loci in a population that has more than one allele; Σ-alleles, the total number of different alleles observed across all loci in a population.

TA B L E 3
Analysis of genic variation statistics for all loci according to Nei (1987) developed for all five mountain populations.
Source of variation d.f.

| DISCUSS ION
The possibility of comparing the genetic variation of R. alpinus with other Rumex species is limited due to the low number of species examined for SSR marker diversity. Indeed, the physiology and ecology of R. alpinus have been studied (Hujerová et al., 2013;Jungová et al., 2022;Řičařová, 2011) much more than genetic variability, with the only published genetic work being on Rumex bucephalophorus subsp. canariensis (Viruel et al., 2015). Unfortunately, Rumex bucephalophorus is an annual Mediterranean plant.
This is the first study on genetic variability and population struc-  (Bohner, 2005;Rehder, 1982), including the Krkonoše Mountains (Červenková & Münzbergová, 2009;Pyšek et al., 2012;Št'astná et al., 2010). This is evident from the pairwise differences, which showed that population structure reflected a pattern of isolation by distance associated with human dispersal in the past (Kopecký, 1973;Kubát, 1990;Lokvenc, 1978 However, based on historical sources (Hendrych, 2001;Lokvenc, 2007), it is known that botanist Caspar Schwenckfelt (1607) in his botanic book Scite aus dem botanischen Teil des Buches did not mention the very conspicuous herb R. alpinus, and such a large plant cannot be overlooked (Hendrych, 2001). The first historical record that supported the allochthonous origin of R. alpinus, but with a different idea of its introduction, is mentioned only by Wimmer in 1844, who described the findings of these plants in mountain huts (Hendrych, 2001 colonists from Styria, whether they were lumberjacks (Klimeš, 2011) or raftsmen (Smrčka, 2016).  (Nei, 1978) for 31 populations of Rumex alpinus in the European Mountains. Bootstrap values of the consensus tree are given in branches. The length of the branches is proportional to the genetic distance. Numbers and the name of populations see in Tables 1, 2. The colors correspond to the colors of the mountains in the Figure 6a. Genton et al., 2005;Hardesty et al., 2012). In the localities, there may have been a drastic reduction in the number of individuals, inbreeding, and thus allele loss as the population settled into the new territory (Wright, 1931;Maron et al., 2004;Keshavarzi & Mosaferi, 2019).
On the other hand, a founder effect could have occurred there when a new territory was settled, and an individual with a unique allele was introduced Matesanz et al., 2014;Oduor et al., 2016). There could have also been an accumulation of mutations or hybridization with the related species Rumex species (Kubát, 1990;Rechinger, 1957;Št'astná et al., 2010;Stehlik, 2002).
In addition, the Hardy-Weinberg equilibrium results showed the Czech Mountains, especially in the Eagle Mountains, no genetic variation at several loci. It is assumed that the population in Eagle Mountains is highly inbred (Šurinová et al., 2018), and the population consisted only of clones (reproduced via rhizomes in falanga habit Klimeš et al., 1993), contributing to the opinion that this species is nonoriginal and is, therefore, a secondary occurrence (Hollingsworth & Bailey, 2000). Obtained results confirmed the previously published hypothesis that R. alpinus was spread mainly by human distribution from small sources of populations in the Middle Ages (Kopecký, 1973;Lokvenc, 2007 (aqua). The x-axis shows the allele frequencies grouped into categories of 0.1, while the y-axis indicates the percentage of alleles in each frequency category. The red lines on the plot denote populations that exhibit a mode shift in the frequency distribution, which is a hallmark of the bottleneck effect. Raycheva & Dimitrova, 2007;Stachurska-Swakoń, 2008)-was higher compared to the nonnative populations in the Czech Mountains. These findings, as reported by Amsellem et al. (2000), support the conclusion that R. alpinus is indeed a nonnative species in the Czech Mountains.
The variation in the Balkan and Carpathian populations suggests that a single introduction from one native-range population is unlikely. In contrast, the great diversity and the high interpopulation differentiation found in Carpathian populations indicated more native sources.

| CON CLUS ION
The genetic variability and population structure of the weedy plant Data curation (equal); software (equal); writing -original draft (equal).

ACK N OWLED G M ENTS
The authors are very grateful to Professor Pavel Klimeš (Horní Maršov, Veselý výlet) for his valuable information about the Alpine colonists.

CO N FLI C T O F I NTER E S T S TATEM ENT
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are in supporting information, and they will be available at time of publication.